Liquid Formalation of a VEGF Antagonist

ABSTRACT

The present invention relates to liquid pharmaceutical compositions of a VEGF antagonist for intravitreal administration comprising a histidine buffer, an inorganic salt, a carbohydrate and a polysorbate.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation of U.S. application Ser. No.16/072,638, filed on Jul. 25, 2018, which is a United States NationalPhase Application of International Application No. PCT.EP2017/051662,filed on Jan. 26, 2017.

FIELD OF THE INVENTION

The present invention relates to liquid pharmaceutical compositions of aVEGF antagonist for intravitreal administration comprising a histidinecontaining buffer, an inorganic salt, a carbohydrate and a polysorbate.

BACKGROUND OF THE INVENTION

Vascular endothelial growth factor (VEGF) is a protein that stimulatesvasculogenesis (i.e. de novo formation of new blood vessels) andangiogenesis (i.e. formation of new blood vessels from pre-existingvessels). There are at least six subtypes of VEGF, i.e. VEGF-A, VEGF-B,VEGF-C, VEGF-D, virus VEGF-E and placental VEGF (PIGF). VEGF-A isassociated with increases of vascular permeability and degeneration ofthe extracellular matrix. Four isomers of VEGF-A that arise fromalternative splicing of mRNA have been reported in humans (VEGF121,VEGF165, VEGF184, VEGF206) (Ferrara and Davis Smyth, Endocr Rev, 1997,18:1-22). Further, VEGF110 is produced from VEGF165 by proteasecleavage. VEGF-A binds to receptors VEGFr-1 and VEGFr-2 (Kajdaniuk etal., Endokrynol Pol, 2011, 62(5):444-55; Kajdaniuk et al., EndokrynolPol, 2011, 62(5):456-64).

The specificity of VEGF action for endothelial cells supports a key rolein the process of abnormal blood vessel growth and vascular leakage.Anti-VEGF agents have demonstrated efficacy in reducing choroidalneovascularisation in both animal models and clinical trials (Okamoto etal. (1997) Am J Pathol 151: 281-91; Adamis et al. (1996) ArchOphthalmol, 114: 66-71). Specifically, anti-VEGF antibodies have beenused for the treatment of treatments of intraocular neovasculardisorders.

Currently available anti-VEGF antibodies are bevacizumab andranibizumab. Bevacizumab is a full-length, humanized murine monoclonalantibody that recognizes all isoforms of VEGF. Ranibizumab is the Fabfragment of the humanized murine monoclonal antibody that is used tocreate bevacizumab and has been affinity-matured so that it binds VEGF-Awith a significantly higher affinity than bevacizumab. Ranibizumab andbevacizumab appear to have similar efficacy profiles in the treatment ofneovascular age-related macular degeneration although rare adverseevents seem to occur more often with bevacizumab (Johnson and Sharma,Curr Opin Ophthalmol, 2013, 24(3):205-12).

Another class of VEGF antagonists is represented by fusion proteins ofparts of the VEGF receptors and the Fc portion of human immunoglobulins.In particular, aflibercept, marketed under the name Eylea®, is arecombinant fusion protein consisting of the VEGF binding portion fromthe extracellular domains of human VEGF receptors 1 and 2 that are fusedto the Fc portion of the human IgG1 immunoglobulin. It is approved forthe treatment of wet macular degeneration and some further oculardiseases.

For medical purposes stable pharmaceutical compositions are of greatinterest, in particular ready-to-use solutions which require nodissolution or reconstitution before use. A main problem of such aliquid composition is a decreasing content of the active ingredient dueto the formation of insoluble particles during repeated freeze/thawcycles during manufacturing or proteins being degraded and formingdegradation products during long-term storage.

WO 2006/104852A2 discloses liquid pharmaceutical formulations ofaflibercept for subcutaneous or intravenous delivery which comprise ahistidine buffer, sodium chloride, sucrose and polysorbate 20.

In particular for pharmaceutical compositions which are intended to bedelivered to the eye, such as pharmaceutical compositions forintravitreal injections, it is important to keep the amount of insolubleparticles at a minimum level, since particles may cause irritation orinflammation when injected into the eye.

US 2015/157709 A1 and US 2015/182623 A1 disclose formulations comprisinga VEGF antagonist and anti-PDGF aptamer which are suitable forophthalmological use. These formulations comprise a buffer with pH 5.0to 8.0 and a tonicity modifier.

WO 2007/149334 A2 describes liquid pharmaceutical compositions ofaflibercept comprising a sodium phosphate buffer, sodium chloride,sucrose and polysorbate 20 which formulations are suitable forophthalmic use.

WO 2015/071348 A1 discloses liquid pharmaceutical formulations ofranibizumab for intravitreal injection comprising a buffer, a non-ionicsurfactant, and, optionally, an inorganic salt, wherein the compositiondoes not contain saccharides.

Nevertheless, there is still a need for a pharmaceutical compositionwhich has a low protein aggregate content and is therefore suitable forintravitreal injection and which is stable in liquid form. Preferably,such a composition is suitable for the treatment of AMD and formulatedin a prefilled syringe.

SUMMARY OF THE INVENTION

The inventors found that a liquid composition comprising a histidinebuffer, a non-ionic surfactant, a VEGF antagonist, an inorganic salt anda carbohydrate has a surprisingly low level of particles and istherefore particularly suitable for intravitreal injection and thetreatment of neovascular intraocular diseases.

A further advantage of the liquid pharmaceutical composition used in thepresent invention is that it does not require a lyophilisation step andis thus produced in a shorter time and with reduced costs. Anotheradvantage is that the composition has a pH in the range of 6.0 to 6.5,i.e. a pH close to the physiological pH.

The object of the present invention is solved by the subject-matter ofthe independent claims. Preferred embodiments are apparent from thedependent claims.

Accordingly, in one embodiment the present invention provides a liquidpharmaceutical composition for use in the treatment of an intraocularneovascular disease comprising

-   -   a) a histidine containing buffer,    -   b) a non-ionic surfactant,    -   c) a VEGF antagonist,    -   d) an inorganic salt, and    -   e) a carbohydrate.

The pH of the composition may be between 6.0 and 6.5, preferably between6.2 and 6.5. Also preferably, the pH is 6.2 or 6.5.

The histidine-containing buffer may be L-histidine/histidinehydrochloride and/or may be present in a concentration of from 1 mM to40 mM, preferably of 10 mM.

The non-ionic surfactant may be polysorbate 20 and/or may be present ina concentration of from 0.01 to 0.08% (w/v), preferably of 0.03% (w/v).

The inorganic salt may be NaCl and/or may be present in a concentrationof from 20 to 100 mM, preferably of 40 mM.

The VEGF antagonist may be an anti-VEGF antibody or an antigen-bindingfragment of such antibody or a VEGF receptor fusion protein, preferablyit may be aflibercept or ranibizumab.

The VEGF antagonist may be present in a concentration of 6 to 45 mg/ml.

The carbohydrate may be sucrose and/or may be present in a concentrationof 3-20% (w/v), preferably of 5% (w/v).

The present invention also relates to a liquid pharmaceuticalcomposition for use in the treatment of an intraocular neovasculardisease consisting of histidine hydrochloride/L-histidine, polysorbate20, NaCl, aflibercept, sucrose and water and having a pH of 6.2 or 6.5.

The present invention also relates to a liquid pharmaceuticalcomposition for use in the treatment of an intraocular neovasculardisease consisting of histidine hydrochloride/L-histidine, polysorbate20, NaCl, aflibercept, sucrose and water and having a pH of 6.2.

The present invention also relates to a liquid pharmaceuticalcomposition for use in the treatment of an intraocular neovasculardisease consisting of histidine hydrochloride/L-histidine, polysorbate20, NaCl, aflibercept, sucrose and water and having a pH of 6.5.

Preferably, the liquid pharmaceutical composition consists of 10 mMhistidine hydrochloride/L-histidine, 0.03% polysorbate 20 (w/v), 40 mMNaCl, 40 mg/ml aflibercept, 5% sucrose and water and has a pH of 6.2 or6.5.

Also preferably, the liquid pharmaceutical composition consists of 10 mMhistidine hydrochloride/L-histidine, 0.03% polysorbate 20 (w/v), 40 mMNaCl, 40 mg/ml aflibercept, 5% sucrose and water and has a pH of 6.2.

Also preferably, the liquid pharmaceutical composition consists of 10 mMhistidine hydrochloride/L-histidine, 0.03% polysorbate 20 (w/v), 40 mMNaCl, 40 mg/ml aflibercept, 5% sucrose and water and has a pH of 6.5.

The present invention also relates to a liquid pharmaceuticalcomposition consisting of 10 mM histidine hydrochloride/L-histidine,0.03% polysorbate 20 (w/v), 40 mM NaCl, 40 mg/ml aflibercept, 5% sucroseand water and having a pH of 6.2 or 6.5.

The present invention also relates to a liquid pharmaceuticalcomposition consisting of 10 mM histidine hydrochloride/L-histidine,0.03% polysorbate 20 (w/v), 40 mM NaCl, 40 mg/ml aflibercept, 5% sucroseand water and having a pH of 6.2.

The present invention also relates to a liquid pharmaceuticalcomposition consisting of 10 mM histidine hydrochloride/L-histidine,0.03% polysorbate 20 (w/v), 40 mM NaCl, 40 mg/ml aflibercept, 5% sucroseand water and having a pH of 6.5.

The present invention also relates to a liquid pharmaceuticalcomposition consisting of 10 mM histidine hydrochloride/L-histidine,0.03% polysorbate 20 (w/v), 40 mM NaCl, a recombinant protein, 5%sucrose and water and having a pH of 6.2 or 6.5.

The present invention also relates to a liquid pharmaceuticalcomposition consisting of 10 mM histidine hydrochloride/L-histidine,0.03% polysorbate 20 (w/v), 40 mM NaCl, a recombinant protein, 5%sucrose and water and having a pH of 6.2.

The present invention also relates to a liquid pharmaceuticalcomposition consisting of 10 mM histidine hydrochloride/L-histidine,0.03% polysorbate 20 (w/v), 40 mM NaCl, a recombinant protein, 5%sucrose and water and having a pH of 6.5.

The intraocular neovascular disease may be age-related maculardegeneration (AMD), visual impairment due to diabetic macular oedema(DME), visual impairment due to macular oedema secondary to retinal veinocclusion (branch RVO or central RVO), or visual impairment due tochoroidal neovascularisation (CNV) secondary to pathologic myopia.

The present invention also relates to a prefilled syringe containing thepharmaceutical composition as defined herein.

BRIEF DESCRIPTION OF THE DRAWINGS

Exemplary embodiments of the invention will be described in thefollowing with reference to the following drawings:

FIG. 1A: Detection of high molecular weight species by size exclusionchromatography with samples F1 to F7_subjected to stress conditions(three freeze/thaw cycles or shaking for seven days).

FIG. 1B: Detection of high molecular weight species by size exclusionchromatography with samples F1 to F7_stored at 5° C. for one or threemonths.

FIG. 1C: Detection of high molecular weight species by size exclusionchromatography with samples F1 to F7_stored at 25° C./60% relativehumidity for two weeks, one, two or three months.

FIG. 1D: Detection of high molecular weight species by size exclusionchromatography with samples F1 to F7 stored at 40° C./75% relativehumidity for two weeks, one, two or three months.

FIG. 2: Analysis of protein fragmentation in samples F3, F4, F5 and F7after storage for three months at 25° C./60% relative humidity or 40°C./75% relative humidity by SDS-PAGE

FIG. 3: Non-reduced SDS-PAGE of the samples S6 and S2 incubated forthree months at 40° C./75% relative humidity

FIG. 4: Non-reduced SDS-PAGE of the samples (a) to (d) shown in Table 9after three months incubation at 40° C./75% relative humidity

FIG. 5: Reduced SDS-PAGE of the samples (a) to (d) shown in table 9after three months incubation at 40° C./75% relative humidity

DETAILED DESCRIPTION OF THE INVENTION

The present invention as illustratively described in the following maysuitably be practiced in the absence of any element or elements,limitation or limitations, not specifically disclosed herein.

The present invention will be described with respect to particularembodiments, but the invention is not limited thereto, but only by theclaims.

Where the term “comprising” is used in the present description andclaims, it does not exclude other elements. For the purposes of thepresent invention, the term “consisting of” is considered to be apreferred embodiment of the term “comprising”. If hereinafter a group isdefined to comprise at least a certain number of embodiments, this isalso to be understood to disclose a group which preferably consists onlyof these embodiments.

For the purposes of the present invention, the term “obtained” isconsidered to be a preferred embodiment of the term “obtainable”. Ifhereinafter e.g. a cell or organism is defined to be obtainable by aspecific method, this is also to be understood to disclose a cell ororganism which is obtained by this method.

Where an indefinite or definite article is used when referring to asingular noun, e.g. “a”, “an” or “the”, this includes a plural of thatnoun unless something else is specifically stated.

The term “pharmaceutical composition” as used herein refers to anycomposition comprising a chemical substance or active ingredient whichcomposition is intended for use in the medical cure, treatment, orprevention of disease and which is in such a form as to permit theactive ingredient to be effective. In particular, a pharmaceuticalcomposition does not contain excipients which are unacceptably toxic toa subject to which the composition is to be administered. Thepharmaceutical compositions are sterile, i.e. aseptic and free from allliving microorganisms and their spores. The pharmaceutical compositionused in the present invention is liquid and stable.

In a “liquid composition” the pharmaceutically active agent, e.g. theVEGF antagonist, can be combined with a variety of excipients to ensurea stable active medication following storage. The liquid pharmaceuticalcomposition used in the invention is at no point lyophilised, i.e. theproduction method does not contain a lyophilisation step and thecomposition is not lyophilised for storage. Liquid compositions can bestored in vials, IV bags, ampoules, cartridges, and prefilled orready-to-use syringes.

A “stable” liquid composition is one in which the VEGF antagonistcontained therein essentially retains its physical stability and/orchemical stability and/or biological activity upon storage for a certainperiod. Preferably, the composition essentially retains upon storage itsphysical and chemical stability, as well as its biological activity.Various analytical techniques for measuring protein stability areavailable in the art and are reviewed, for example, in Peptide andProtein Drug Delivery, 247-301, Vincent Lee Ed, Marcel Dekker, Inc, NewYork, N.Y., Pubs (1991) and Jones, Adv Drug Delivery Rev, 1993,10:29-90. For example, stability can be measured at a selectedtemperature for a selected time period. Stability can be evaluatedqualitatively and/or quantitatively in a variety of different ways,including evaluation of aggregate formation (for example using sizeexclusion chromatography, by measuring turbidity, and/or by visualinspection), by assessing charge heterogeneity using cation exchangechromatography or capillary zone electrophoresis, amino-terminal orcarboxy-terminal sequence analysis, mass spectrometric analysis,SDS-PAGE analysis to detect aggregated or fragmented molecules, peptidemap (for example tryptic or LYS-C) analysis, evaluating biologicalactivity or binding of the antagonist, etc.

Preferably, the pharmaceutical composition is stable at a temperature ofabout 40° C. for at least 1 to 2 weeks, and/or is stable at atemperature of about 5° C. for at least 3 months, and/or is stable at atemperature of about 25° C. for at least two weeks or one month.Furthermore, the formulation is preferably stable following freezing(to, e.g., −20° C.) and thawing of the formulation at 25° C. asdescribed in the examples herein, for example following 1, 2, 3 or 4cycles of freezing and thawing.

For example, in the pharmaceutical composition used in the presentinvention the percentage of high molecular weight species relative tothe total amount of the VEGF antagonist as measured by size exclusionchromatography is not more than 4%, preferably not more than 3.5% or3.25%, more preferably not more than 3.0% or 2.75% and most preferablynot more than 2.5% after storage of the composition at 5° C. for 3months.

A “buffer” is an aqueous solution consisting of a mixture of a weak acidand its conjugate base or vice versa which resists changes in its pH andtherefore keeps the pH at a nearly constant value. The buffer of thepresent invention preferably has a pH in the range from about 6.0 toabout 7.0, preferably from about 6.1 to about 6.8, more preferably fromabout 6.0 to 6.5, even more preferably from about 6.2 to 6.5 and mostpreferably has a pH of about 6.2 or 6.5.

The buffer used in the present invention is a histidine-containingbuffer. Preferably, the histidine-containing buffer is the only bufferpresent in the liquid formulation of the present invention.

The terms “histidine-containing buffer” and “histidine buffer” are usedinterchangeably herein and refer to a buffer comprising histidine.Examples of histidine buffers include histidine chloride, histidinehydrochloride, histidine acetate, histidine phosphate, and histidinesulphate. The preferred histidine buffer of the invention furthercomprises L-histidine. Even more preferably the histidine buffer of theinvention comprises histidine hydrochloride, most preferably itcomprises histidine hydrochloride and L-histidine. Preferably, thehistidine buffer or histidine hydrochloride buffer or histidinehydrochloride/L-histidine buffer has a pH in the range from about 6.0 toabout 7.0, preferably from about 6.1 to about 6.8, more preferably fromabout 6.0 to 6.5, even more preferably from about 6.2 to 6.5 and mostpreferably has a pH of about 6.2 or 6.5.

In a particular preferred embodiment, the histidine-containing buffercomprises histidine hydrochloride/L-histidine in a concentration in therange of 1 mM to 40 mM, preferably of 2 mM to 35 mM, more preferably of3 mM to 30 mM, even more preferably of 5 mM to 20 mM and most preferablyof 8 mM to 15 mM.

In another particular preferred embodiment the buffer is histidinehydrochloride/L-histidine with a concentration of 10 mM.

In another particular preferred embodiment the buffer is histidinehydrochloride/L-histidine with a concentration of 10 mM and with a pH of6.2 or 6.5.

The pharmaceutical compositions of the present invention are preferablyprepared by dissolving L-histidine, L-histidine-HCl, the carbohydrate,preferably sucrose, and the inorganic salt, preferably sodium chloride,in water before adding the non-ionic surfactant, preferably polysorbate20 and then adding the VEGF antagonist.

A “surfactant” as used herein refers to an amphiphilic compound, i.e. acompound containing both hydrophobic groups and hydrophilic groups whichlowers the surface tension (or interfacial tension) between two liquidsor between a liquid and a solid. A “non-ionic surfactant” has no chargedgroups in its head. The formation of insoluble particles duringfreeze/thaw cycles of antibody-containing compositions can be remarkablyinhibited by addition of surfactants. Examples of “non-ionicsurfactants” include e.g. polyoxyethylene glycol alkyl ethers, such asoctaethylene glycol monododecyl ether, pentaethylene glycol monododecylether; polyoxypropylene glycol alkyl ethers; glucoside alkyl ethers,such as decyl glucoside, lauryl glucoside, octyl glucoside;polyoxyethylene glycol octylphenol ethers, such as triton X-100;polyoxyethylene glycol alkylphenol ethers, such as nonoxynol-9; glycerolalkyl esters, such as glyceryl laurate; polyoxyethylene glycol sorbitanalkyl esters, such as polysorbate; sorbitan alkyl esters, such as spans;cocamide MEA, cocamide DEA, dodecyldimethylamine oxide; block copolymersof polyethylene glycol and polypropylene glycol, such as poloxamers; andpolyethoxylated tallow amine (POEA). The pharmaceutical compositions ofthe present invention can contain one or more of these surfactants incombination.

Preferred non-ionic surfactants for use in the pharmaceuticalcompositions of the present invention are polysorbates such aspolysorbate 20, 40, 60 or 80, and especially polysorbate 20 (i.e. Tween20).

The concentration of the non-ionic surfactant is in the range of 0.01 to0.08% (w/v), preferably in the range of 0.015 to 0.06% (w/v), morepreferably in the range of 0.02 to 0.04% (w/v) and most preferably it is0.03% (w/v), relative to the total volume of the composition.

In a preferred embodiment, the non-ionic surfactant is polysorbate 20with a concentration in the range of 0.015 to 0.06% (w/v), morepreferably in the range of 0.02 to 0.04% (w/v) and most preferably it is0.03% (w/v), relative to the total volume of the composition.

In a particularly preferred embodiment the non-ionic surfactant ispolysorbate 20 with a concentration of 0.03% (w/v), relative to thetotal volume of the composition.

Herein, an “inorganic salt” refers to a ionic compound which hasosmoregulatory properties. An inorganic salt such as sodium chloride(NaCl) can dissociate in solution into its constituent ions, i.e. NaCldissociates into Na⁺ and Cl⁻ ions, which both affect the osmoticpressure, i.e. the osmolality, of the solution. Preferred inorganicsalts for use in the pharmaceutical formulation of the present inventionare potassium chloride, calcium chloride, sodium chloride, sodiumphosphate, potassium phosphate and sodium bicarbonate. Preferably theinorganic salt is a sodium salt, more preferably it is sodium chloride(NaCl).

The concentration of the inorganic salt in the pharmaceuticalcomposition used in the present invention is preferably in the range of20 to 100 mM, more preferably in the range of 25 to 80 mM, even morepreferably the inorganic salt has a concentration in the range of 30 to60 mM or 35 to 45 mM, and most preferably the concentration is 40 mM.

In a particular preferred embodiment, the inorganic salt is NaCl with aconcentration in the range of 20 to 100 mM, more preferably in the rangeof 25 to 80 mM, even more preferably the inorganic salt has aconcentration in the range of 30 to 60 mM or 35 to 45 mM, and mostpreferably the concentration is 40 mM.

In a most preferred embodiment the inorganic salt is NaCl with aconcentration of 40 mM.

In a further embodiment the pharmaceutical composition comprises aninorganic salt, preferably NaCl, preferably in a concentration of 40 mM,polysorbate 20 in a concentration of 0.03% (w/v), sucrose in aconcentration of 5% (w/v) and a histidine hydrochloride/L-histidinebuffer with a concentration of 10 mM and a pH of 6.5, or a histidinehydrochloride/L-histidine buffer with concentration of 10 mM and a pH of6.2.

The term “VEGF antagonist” refers to a molecule which specificallyinteracts with VEGF and inhibits one or more of its biologicalactivities, e.g. its mitogenic, angiogenic and/or vascular permeabilityactivity. It is intended to include both anti-VEGF antibodies andantigen-binding fragments thereof and non-antibody VEGF antagonists.

Non-antibody VEGF antagonists include aflibercept, pegaptanib andantibody mimetics. Preferably, the non-antibody VEGF antagonist isaflibercept. Aflibercept which is presently marketed under the nameEylea® and which is also known as VEGF-trap is a recombinant humansoluble VEGF receptor fusion protein in which the secondimmunoglobulin-like domain of VEGF receptor 1 and the thirdimmunoglobulin-like domain of VEGF receptor 2 are fused to the Fcportion of human IgG1 (Holash et al. (2002) Proc. Natl. Acad. Sci. USA99(17): 11393-11398; WO 00/75319 A1). The CAS number of aflibercept is862111-32-8. It has received a marketing authorization for the treatmentof wet age-related macular degeneration, visual impairment due todiabetic macular oedema (DME) and diabetic retinopathy in patients withdiabetic macular edema. The present commercial aflibercept formulationcontains sodium phosphate, sodium chloride, polysorbate 20, sucrose andwater for injection and is supplied in a concentration of 40 mg/ml.

Pegaptanib which is presently marketed under the name Macugen® is apegylated anti-vascular endothelial growth factor (VEGF) aptamer (Bellet al. (1999) In Vitro Cell Dev Biol Anim. 35(9): 533-42). Antibodymimetics which are VEGF antagonists include binding proteins comprisingan ankyrin repeat domain that binds VEGF and inhibits its binding to thereceptor, such as DARPin® MP0112 (see also WO 2010/060748 and WO2011/135067).

The term “antibody” or “immunoglobulin” is used herein in the broadestsense and includes full length antibodies, genetically engineeredantibodies, recombinant antibodies, multivalent antibodies, monoclonalantibodies, polyclonal antibodies, bispecific antibodies, multispecificantibodies, chimeric antibodies, humanized antibodies, fully humanantibodies, as well as fragments of such antibodies as long as theyremain functional and exhibit the desired biological activity. The“Biological activity” of an antibody refers to the ability of theantibody to bind to antigen and result in a biological response whichcan be measured in vitro or in vivo.

A full length antibody comprises an antigen-binding variable region ofthe light (V_(L)) and heavy chain (V_(H)), a light chain constant region(C_(L)) and heavy chain constant domains C_(H)1, C_(H)2 and C_(H)3.

The term “antibody fragment” or “antigen-binding fragment” is usedherein in the broadest sense and comprises a portion of a full lengthantibody, preferably comprising the antigen-binding or variable regionthereof. An antibody fragment retains the original specificity of theparent immunoglobulin. Examples of antibody fragments include, e.g.,Fab, Fab′, F(ab′)₂, and Fv fragments, diabodies, linear antibodies,single-chain antibody molecules, and multispecific antibodies formedfrom antibody fragment(s). Preferably, the antibody fragment is a Fabfragment.

A “monoclonal antibody” is an antibody that is specific for a singleepitope of an antigen, i.e. directed against a single determinant on anantigen. Methods for producing monoclonal antibodies are known to theperson skilled in the art.

The term “recombinant antibody” refers to all antibodies prepared,expressed, created or isolated by recombinant means, such as antibodiesisolated from a transgenic host cell, such as e.g. a NS0 or CHO cell, orfrom an animal transgenic for immunoglobulin genes, or antibodiesexpressed using recombinant expression vectors transfected into a hostcell, such as e.g. SP 2/0 mouse myeloma cells.

A “humanised antibody” is a human antibody wherein the antigen bindingportion (CDR) is derived from non-human species, such as a mouse, andthus has a different specificity compared to the parent immunoglobulin.The CDR protein sequences can be modified to increase their similaritiesto antibody variants produced naturally in humans.

The term “anti-VEGF antibody” refers to an antibody or antibody fragmentsuch as a Fab or a scFV fragment that specifically binds to VEGF andinhibits one or more of its biological activities, e.g. its mitogenic,angiogenic and/or vascular permeability activity. Anti-VEGF antibodiesact, e.g., by interfering with the binding of VEGF to a cellularreceptor, by interfering with vascular endothelial cell activation afterVEGF binding to a cellular receptor, or by killing cells activated byVEGF. Anti-VEGF antibodies include, e.g., antibodies A4.6.1,bevacizumab, ranibizumab, G6, B20, 2C3, and others as described in, forexample, WO 98/45331, US 2003/0190317, U.S. Pat. Nos. 6,582,959,6,703,020, WO 98/45332, WO 96/30046, WO 94/10202, WO 2005/044853, EP 0666 868 B1, WO 2009/155724 and Popkov et al. (2004) J. Immunol. Meth.288: 149-64. Preferably, the anti-VEGF antibody or antigen-bindingfragment thereof present in the pharmaceutical composition used in thepresent invention is ranibizumab or bevacizumab. Most preferably, it isranibizumab or an antigen-binding fragment thereof.

“Ranibizumab” is a humanised monoclonal Fab fragment directed againstVEGF-A having the light and heavy chain variable domain sequences ofY0317 as described in SEQ ID Nos. 115 and 116 of WO 98/45331 and Chen etal. (1999) J. Mol. Biol. 293: 865-81. The CAS number of ranibizumab is347396-82-1. Ranibizumab inhibits endothelial cell proliferation andneovascularisation and has been approved for the treatment ofneovascular (wet) age-related macular degeneration (AMD), the treatmentof visual impairment due to diabetic macular oedema (DME), the treatmentof visual impairment due to macular oedema secondary to retinal veinocclusion (branch RVO or central RVO), or treatment of visual impairmentdue to choroidal neovascularisation (CNV) secondary to pathologicmyopia. Ranibizumab is related to bevacizumab and derived from the sameparent mouse antibody as bevacizumab but it is much smaller than theparent molecule and has been affinity matured to provide strongerbinding to VEGF-A. Ranibizumab is produced recombinantly in Escherichiacoli, e.g. as described in WO 98/45331 A2. The present commercialranibizumab formulation contains α,α-trehalose dihydrate, histidinehydrochloride monohydrate, histidine, polysorbate 20 and water forinjection and is supplied in a concentration of 10 mg/ml.

“Bevacizumab” is a full-length, humanized murine monoclonal antibodythat recognizes all isoforms of VEGF and which is the parent antibody ofranibizumab. The CAS number of bevacizumab is 216974-75-3. Bevacizumabinhibits angiogenesis and is presently approved for the treatment ofdifferent cancer types. However, it is also used off-label inophthalmological diseases such as age-related macular degeneration. Thepresent commercial bevacizumab formulation contains α,α-trehalosedihydrate, sodium phosphate, polysorbate 20 and water for injection andis supplied as a concentrate with a concentration of 25 mg/ml.

In one embodiment, the VEGF antagonist is the only pharmacologicallyactive agent within the formulation. In an alternative embodiment, theformulation contains one or more pharmacologically active agents inaddition to the VEGF antagonist. A pharmacologically active agent isable to exert a pharmacological effect when administered to a subject.Preferably, the additional pharmacologically active agent is a PDGFantagonist or an Ang2 antagonist. More preferably, the PDGF antagonistis an anti-PDGF antibody such as rinucumab or an aptamer such as E10030,marketed as Fovista®. Most preferably, the PDGF antagonist is E10030which is described in Green et al. (1996) Biochemistry 35: 14413; U.S.Pat. Nos. 6,207,816; 5,731,144; 5,731,424; and 6,124,449. Also morepreferably, the Ang2 antibody is an anti-Ang2 antibody and mostpreferably it is nesvacumab.

The concentration of the VEGF antagonist in the pharmaceuticalcompositions is typically 5-80 mg/ml, preferably 7-60 mg/ml, morepreferably 8-50 mg/ml and most preferably 10 or 40 mg/ml. If the VEGFantagonist is aflibercept, the concentration of the VEGF antagonist,i.e. aflibercept, is preferably 40 mg/ml. If the VEGF antagonist isranibizumab, the concentration of the VEGF antagonist, i.e. ranibizumab,is preferably 6 or 10 mg/ml.

The term “carbohydrate” refers to an organic compound comprising onlycarbon, hydrogen, and oxygen, usually with a hydrogen:oxygen atom ratioof 2:1 and the empirical formula C_(m)(H₂O)_(n). The term “carbohydrate”includes mono-, di-, oligo- and polysaccharides. Examples ofcarbohydrates include glucose, fructose, galactose, xylose, ribose,sucrose, mannose, lactose, maltose, trehalose, starch, and glycogen.Various other forms of sugars are known, e.g., sugar alcohols such asglycerol, mannitol, sorbitol, and xylitol; sugar acids, e.g. aldonicacids such as ascorbic acid, aldaric acids such as tartaric acid;reducing sugars, e.g. glucose, glyceraldehydes, galactose, lactose, andmaltose; amino sugars, e.g. N-acetylglucosamine, galactosamine,glucosamine, and sialic acid; or sulfoquinovose, a sulphonic acidderivative of glucose.

The pharmaceutical composition used in the present invention may furthercontain diluents, solubilising agents, isotonising agents, excipients,pH-modifiers, soothing agents, buffers, sulphur-containing reducingagents, antioxidants or the like. The pharmaceutical composition used inthe present invention does not contain PEG3350 and/or glycine.

Preferably, the pharmaceutical composition used in the present inventioncontains histidine hydrochloride/L-histidine, polysorbate 20, NaCl,sucrose, water and aflibercept and no further components or activesubstances, i.e. the pharmaceutical composition consists of histidinehydrochloride/L-histidine, polysorbate 20, NaCl, sucrose, water andaflibercept. More preferably, the pharmaceutical composition used in thepresent invention consists of 10 mM histidine hydrochloride/L-histidine,0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose, water and 40mg/ml aflibercept.

Also preferably, the pharmaceutical composition used in the presentinvention contains histidine hydrochloride/L-histidine, polysorbate 20,NaCl, sucrose, water and ranibizumab and no further components or activesubstances, i.e. the pharmaceutical composition consists of histidinehydrochloride/L-histidine, polysorbate 20, NaCl, sucrose, water andranibizumab. More preferably, the pharmaceutical composition used in thepresent invention consists of 10 mM histidine hydrochloride/L-histidine,0.03% (w/v) polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose, water and 10mg/ml ranibizumab.

An “intraocular neovascular disease” is a disease characterized byocular neovascularisation. Examples of intraocular neovascular diseasesinclude, e.g., proliferative retinopathies, choroidal neovascularisation(CNV), age-related macular degeneration (AMD), diabetic and otherischemia-related retinopathies, diabetic macular oedema, pathologicalmyopia, von Hippel-Lindau disease, histoplasmosis of the eye, CentralRetinal Vein Occlusion (CRVO), Branch Retinal Vein Occlusion (BRVO),corneal neovascularisation, and retinal neovascularisation. The term“age-related macular degeneration” refers to a medical condition whichusually affects older adults and results in a loss of vision in thecentre of the visual field (the macula) because of damage to the retina.

If the VEGF antagonist present in the pharmaceutical composition used inthe present invention is aflibercept, the pharmaceutical composition ispreferably for use in the treatment of neovascular (wet) age-relatedmacular degeneration (AMD), visual impairment due to macular oedemasecondary to retinal vein occlusion (branch RVO or central RVO), visualimpairment due to diabetic macular oedema (DME) or visual impairment dueto myopic choroidal neovascularisation (myopic CNV).

If the VEGF antagonist present in the pharmaceutical composition used inthe present invention is ranibizumab, the pharmaceutical composition ispreferably for use in the treatment of neovascular (wet) age-relatedmacular degeneration (AMD), of visual impairment due to diabetic macularedema (DME), of visual impairment due to macular edema secondary toretinal vein occlusion (branch RVO or central RVO) or of visualimpairment due to choroidal neovascularisation (CNV) secondary topathologic myopia (PM).

The term “intravitreal injection” refers to the administration of apharmaceutical composition in which the substance is injected directlyinto the eye. More specifically, the substance is injected into thevitreous humour (also called vitreous body or simply vitreous) which isthe clear gel that fills the space between the lens and the retina ofthe eyeball of humans and other vertebrates.

Pharmaceutical compositions of the present invention can be supplied insealed and sterilized plastic, glass or other suitable containers havinga defined volume such as vials, ampoules or syringes or a large volumesuch as bottles.

It is preferred that the liquid pharmaceutical composition containing aVEGF antagonist, preferably aflibercept or ranibizumab, is supplied in aprefilled syringe. A “ready-to-use syringe” or “prefilled syringe” is asyringe which is supplied in a filled state, i.e. the pharmaceuticalcomposition to be administered is already present in the syringe andready for administration. Prefilled syringes have many benefits comparedto separately provided syringe and vial, such as improved convenience,affordability, accuracy, sterility, and safety. The use of prefilledsyringes results in greater dose precision, in a reduction of thepotential for needle sticks injuries that can occur while drawingmedication from vials, in pre-measured dosage reducing dosing errors dueto the need to reconstituting and/or drawing medication into a syringe,and in less overfilling of the syringe helping to reduce costs byminimising drug waste. The barrel of the pre-filled syringe may be madeof glass or plastic. Preferably, the barrel of the pre-filled syringe ismade of plastic, more preferably of cyclic olefin polymer. Preferably,the barrel of the pre-filled syringe is not coated with silicone.

In a preferred embodiment the pH of the liquid pharmaceuticalcomposition used in the present invention is in the range from about 6.0to about 7.0, preferably from about 6.1 to about 6.8, more preferablyfrom about 6.0 to 6.5, even more preferably from about 6.2 to 6.5 andmost preferably has a pH of about 6.2 or 6.5.

The liquid pharmaceutical composition used in the present invention isto be used in the treatment of an intraocular neovascular disease suchas age-related macular degeneration (AMD), in the treatment of visualimpairment due to diabetic macular oedema (DME), in the treatment ofvisual impairment due to macular oedema secondary to retinal veinocclusion (branch RVO or central RVO), or in the treatment of visualimpairment due to choroidal neovascularisation (CNV) secondary topathologic myopia.

In particular, the invention relates to a liquid pharmaceuticalcomposition for use in the treatment of an intraocular neovasculardisease such as AMD comprising a histidine-containing buffer, anon-ionic surfactant, an inorganic salt, a carbohydrate and a VEGFantagonist.

In one embodiment of the invention the liquid pharmaceutical compositionfor intravitreal administration for use in the treatment of anintraocular neovascular disease such as AMD comprises ahistidine-containing buffer, a non-ionic surfactant, an inorganic salt,a carbohydrate and a VEGF antagonist.

In a preferred embodiment of the invention the liquid pharmaceuticalcomposition for intravitreal administration for use in the treatment ofan intraocular neovascular disease such as AMD comprises ahistidine-containing buffer in a concentration of 1 mM to 40 mM, anon-ionic surfactant in a concentration of 0.01 to 0.08% (w/v), aninorganic salt in a concentration of 20 to 100 mM, a carbohydrate in aconcentration of 3 to 20% (w/v) and a VEGF antagonist.

In another preferred embodiment of the invention the liquidpharmaceutical composition for intravitreal administration for use inthe treatment of an intraocular neovascular disease such as AMDcomprises histidine hydrochloride/L-histidine in a concentration of 1 mMto 40 mM, polysorbate 20 in a concentration of 0.01 to 0.08% (w/v), NaClin a concentration of 20 to 100 mM, sucrose in a concentration of 3 to20% (w/v) and aflibercept.

In another preferred embodiment of the invention the liquidpharmaceutical composition for intravitreal administration for use inthe treatment of an intraocular neovascular disease such as AMDcomprises 10 mM histidine hydrochloride/L-histidine, 0.03% (w/v)polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and aflibercept.

In another preferred embodiment of the invention the liquidpharmaceutical composition for intravitreal administration for use inthe treatment of an intraocular neovascular disease comprises 10 mMhistidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mMNaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept.

In another preferred embodiment of the invention the liquidpharmaceutical composition for intravitreal administration for use inthe treatment of an intraocular neovascular disease consists of 10 mMhistidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mMNaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept.

In another preferred embodiment of the invention the liquidpharmaceutical composition for intravitreal administration for use inthe treatment of an intraocular neovascular disease consists of 10 mMhistidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mMNaCl, 5% (w/v) sucrose and 40 mg/ml aflibercept and has a pH of 6.2 or6.5.

In another preferred embodiment of the invention the liquidpharmaceutical composition for intravitreal administration for use inthe treatment of an intraocular neovascular disease such as AMDcomprises histidine hydrochloride/L-histidine in a concentration of 1 mMto 40 mM, polysorbate 20 in a concentration of 0.01 to 0.08% (w/v), NaClin a concentration of 20 to 100 mM, sucrose in a concentration of 3 to20% (w/v) and ranibizumab.

In another preferred embodiment of the invention the liquidpharmaceutical composition for intravitreal administration for use inthe treatment of an intraocular neovascular disease such as AMDcomprises 10 mM histidine hydrochloride/L-histidine, 0.03% (w/v)polysorbate 20, 40 mM NaCl, 5% (w/v) sucrose and ranibizumab.

In another preferred embodiment of the invention the liquidpharmaceutical composition for intravitreal administration for use inthe treatment of an intraocular neovascular disease comprises 10 mMhistidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mMNaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab.

In another preferred embodiment of the invention the liquidpharmaceutical composition for intravitreal administration for use inthe treatment of an intraocular neovascular disease consists of 10 mMhistidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mMNaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab.

In another preferred embodiment of the invention the liquidpharmaceutical composition for intravitreal administration for use inthe treatment of an intraocular neovascular disease consists of 10 mMhistidine hydrochloride/L-histidine, 0.03% (w/v) polysorbate 20, 40 mMNaCl, 5% (w/v) sucrose and 6 or 10 mg/ml ranibizumab and has a pH of 6.2or 6.5.

Moreover, the invention encompasses the intravitreal administration ofthe liquid pharmaceutical composition of the invention to a subject inan effective amount to treat an intraocular neovascular disease such asAMD. In a preferred embodiment, the liquid pharmaceutical composition ofthe invention for intravitreal administration is present in a prefilledsyringe.

While the invention has been illustrated and described in detail in thedrawings and foregoing description, such illustration and descriptionare to be considered illustrative or exemplary and not restrictive. Theinvention is not limited to the disclosed embodiments. Other variationsto the disclosed embodiments can be understood and effected by thoseskilled in the art in practicing a claimed invention, from a study ofthe drawings, the disclosure, and the dependent claims.

The detailed description is merely exemplary in nature and is notintended to limit application and uses. The following examples furtherillustrate the present invention without, however, limiting the scope ofthe invention thereto. Various changes and modifications can be made bythose skilled in the art on the basis of the description of theinvention, and such changes and modifications are also included in thepresent invention.

EXAMPLES

Examples 1.1 to 1.4 relate to a first set of stability tests performedwith the seven compositions of Table 1 below. Examples 2.1 to 2.6 showthe result of stability tests of samples 2 and 6 as described in Table 2in prefilled syringes. Examples 3.1 to 3.9 relate to a second set ofstability tests performed with the four compositions of Table 9.

Example 1: First Set of Stability Tests Example 1.1: Preparation ofSamples

Different formulations of aflibercept were prepared according to Table1.

TABLE 1 Pharmaceutical compositions tested. Sample 7 corresponds to thecommercially available aflibercept (Eylea ®) formulation. bufferinorganic salt surfactant carbohydrate 1 10 mM sodium citrate, 40 mMNaCl 0.03% PS 20 5% sucrose pH 6.2 2 10 mM sodium citrate, — 0.03% PS 2010% sucrose pH 6.2 3 10 mM L-His/HisHCl; 40 mM NaCl 0.03% PS 20 5%sucrose pH 6.2 4 10 mM L-His/HisHCl; — 0.03% PS 20 10% sucrose pH 6.2 510 mM L-His/HisHCl; 40 mM NaCl 0.03% PS 20 5% sucrose pH 6.5 6 10 mML-His/HisHCl; 150 mM NaCl 0.01% PS 20 — pH 6.2 7 10 mM sodium phosphate;40 mM NaCl 0.03% PS 20 5% sucrose pH 6.2 All pharmaceutical compositionslisted above contained 40 mg/ml of aflibercept.

The pharmaceutical compositions were prepared without aflibercept, whichwas dialyzed into them afterwards. The excipients sucrose, potentiallysodium chloride and the surfactant polysorbate 20 were dissolved in theindicated concentrations of the buffering components L-histidine andL-histidine hydrochloride or tri-sodium citrate and citric acid,respectively.

The ratio of basic and acidic components of the buffer led to a pH of6.2 or 6.5, respectively. The osmolality was determined by GonotecOsmomat 030. All formulations were adjusted to 300 mOsmol/kg+/−20mOsm/kg to reach isotonicity.

Before dialysis the dialysis tubes were hydrated with H₂O. Dialysis ofpooled aflibercept was conducted overnight at 6° C. by regeneratedcellulose membranes with a molecular weight cut off 12 kDa-14 kDa. Afterdialysis the concentration of aflibercept was adjusted to 40 mg/mL+/−2mg/mL by centrifugal filters Vivaspin 6, MWCO 50 kDa and the afliberceptformulations were sterile filtered (0.2 μm PVDF membrane syringefilters) and aseptically filled into pre-sterilized 2 mL FIOLAX type Iglass vials.

Example 1.2: Test Conditions

To identify the most stable formulation the samples were analyzed aftersubjecting them to different conditions.

In one set of experiments, the samples were subjected to differentstress conditions. These stress conditions were chosen to force thechemical and physical degradation pathways of aflibercept and includedthe following conditions:

-   -   a) shaking (samples were shaken with 300 rpm at 25° C. for 7        days), and    -   b) freeze/thaw (samples were frozen and thawed three times        (25° C. to −20° C.) with a rate of ±1° C./min; after each        cooling/heating step the temperature (25° C. and −20° C.        respectively) was kept constant for 10 minutes).

In another set of experiments, the samples were stored at a temperatureof 5° C. for one or three months or at temperatures of 25° C. and 60%relative humidity or 40° C. and 75% relative humidity for two weeks,one, two or three months.

After the samples had been subjected to the different conditions asoutlined above, aliquots were taken and subjected to analysis, e.g. bysodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) andsize exclusion chromatography.

Example 1.3: Analysis by Size Exclusion Chromatography

Size exclusion chromatography was employed to detect high molecularweight species (HMWS), i.e. aggregates of aflibercept.

Size exclusion chromatography (SEC) uses porous particles to separatemolecules of different sizes. It is generally used to separatebiological molecules according to their molecular mass and shape indiluted solution. The stationary phase consists of spherical porousparticles of controlled pore size through which biomolecules diffuse todifferent extents based on differences in their molecular sizes. Smallmolecules diffuse freely into the pores and their movement through thecolumn is retarded, whereas large molecules are unable to enter thepores and are therefore eluted earlier. Hence, molecules are separatedin order of decreasing molecular mass, with the largest moleculeseluting from the column first.

The samples of the stability program were diluted with SEC eluent to afinal concentration of 0.5 mg/mL and stored in HPLC vials at 6° C. untilSEC measurement.

The conditions for size exclusion chromatography were as follows:

Column: TSKgel G3000SWXL

Flow rate: 1.0 mL/min, isocratic elution

Column temperature: 25° C.

Autosampler temperature: 6° C.

Detection: UV 214 nm/280 nm

Injection volume: 20 μL (c=0.5 mg/mL)

Eluent: 0.02 M sodium phosphate with 0.8M NaCl, pH 6.0

Run time: 20 minutes

The results of the detection at 214 nm were used for the evaluation. Themain peak and all other sample specific peaks with a signal to noiseratio of ≥10 were evaluated. The areas of particular peaks e.g.aggregated species were compared to the sum of all sample specific peakswith S/N≥10 giving the relative ratio.

The results of this analysis are shown in FIG. 1. When stored for threemonths at 25° C. or 40° C. formulations F3 and F5 comprising 10 mML-histidine/histidine hydrochloride, 40 mM NaCl, 5% sucrose and 0.03%polysorbate 20 and having a pH of 6.2 and 6.5, respectively, showed thelowest amounts of high molecular weight species, i.e. of aggregatedprotein. In particular, the amount of high molecular weight species informulations F3 and F5 was lower than in the commercially availableaflibercept (Eylea®) formulation. Hence, these formulations can beconsidered more stable than the commercially available aflibercept(Eylea®) formulation.

Example 1.4: SDS PAGE

By SDS-PAGE physical modifications like fragmentation andoligomerisation of aflibercept in the different formulations 1-7 ofTable 1 were determined.

The SDS-PAGE was performed under reducing conditions. Samples werediluted to 0.4 mg/ml with water and further diluted to 0.2 mg/ml withreducing SDS sample buffer. The samples were incubated at 95° C. for 5min. The sample wells were washed with running buffer prior toapplication of the samples (n=2). After the run the gel was washed withwater and dyed with Coomassie overnight. After discoloration the gel wasscanned and analyzed using QuantityOne Software.

The running conditions were as follows:

-   -   voltage: 125 V    -   current: 35 mA    -   power: 5 W    -   time: 110 min

The results of the SDS PAGE analysis are shown in FIG. 2.

In the SDS-PAGE analysis of all samples incubated for three months at25° C. or 40° C. bands representing fragments of aflibercept werevisible. The lowest amount of fragments was detectable for formulationF5 which contains 10 mM L-histidine/histidine hydrochloride, pH 6.5, 40mM NaCl, 5% sucrose and 0.03% polysorbate 20.

Example 2: Stability of Aflibercept Formulations in Prefilled SyringesExample 2.1: Preparation of Pre-Filled Syringes Containing theAflibercept Formulation

165 μl of a solution containing 40 mg/ml of the VEGF antagonistaflibercept and 10 mM histidine buffer, 40 mM sodium chloride, 5% (w/v)sucrose, 0.03% (w/v) polysorbate 20, pH 6.2 was filled into the syringesas listed in Table 2:

TABLE 2 Silicone Syringe Syringe Syringe level Stopper No. size barreltype [mg] coating 2 1.0 ml Borosilicate Luer cone 0.16 Fluoropolymerglass (baked-on) (Flurotec) 6 1.0 mL Cycloolefin Luer cone NoFluoropolymer polymer silicone (Flurotec) The syringes as listed inTable 2 were incubated at 5° C., 25° C/ 60% relative humidity and 40°C./75% relative humidity for one month and 3 months.

Afterwards, the samples were analyzed by UV-Vis for proteinconcentration, by size exclusion chromatography (SEC) and asymmetricflow field-flow fractionation (AF4) for the presence of high molecularweight species (HMWS), by non-reduced sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) for the presence offragments and HMWS, by reduced peptide mapping for the presence ofmethionine oxidation and deamidation. Isoelectric focusing (IEF) wasused to analyze samples for chemical modifications which results incharge variants of aflibercept. Also pH was monitored within the wholeincubation period.

During the complete stability program in all samples no significantchange as well in protein concentration (spectrophotometricquantification at 280 nm; n=3) and pH (n=2) was detected.

Example 2.2: AF4 Analysis of the Aflibercept Formulation Stored inPrefilled Syringes

The asymmetric flow field flow fractionation (AF4) is a technique toidentify and quantify higher molecular weight species of afliberceptbased on their size. This separation is obtained by the difference inmobility (diffusion coefficient) in the flow field induced by the liquidflow across the channel. In combination with MALS (multi angle lightscattering) and UV (280 nm) as concentration-dependent detector, theaflibercept aggregates can be characterized and quantified.

20 μg aflibercept were loaded onto a 15.5 cm separation channel 15.5 cm(short channel) combined with a W490 separation spacer (both WyattTechnology) and a PLGC 10 kD SC-5 Membrane (Millipore). The protein waseluted using 0.1 M sodium phosphate (pH 6.0) and 0.02% sodium azideaccording to elution conditions shown in Table 3 representing the crossflow and focus flow during the separation (channel flow: 0.8 mL/min).

Eluted species were detected at a wavelength of 280 nm and displayed ona graph showing the concentration of the eluted species vs. time. Theelution profile showed a main peak with the non-aggregated protein andsome further peaks of the protein representing higher molecular weightforms of the protein. The corresponding molecular weights werecalculated with a MALLS detector.

TABLE 3 Delta t Time X_(Start) X_(End) FF Step [min] [min] Mode [mL/min][mL/min] [mL/min] 1 4.0 4.0 Elution 1.5 1.5 — 2 1.0 5.0 Focus — — 2.0 32.0 7.0 Focus + — — 2.0 Inj. 4 1.0 8.0 Focus — — 2.0 5 32.0 40.0 Elution1.5 1.5 — 6 10.0 50.0 Elution 1.5 0.2 — 7 10.0 60.0 Elution 0.2 0.2 — 810.0 70.0 Elution + 0.2 0.0 — Inj. 9 10.0 80.0 Elution + 0.0 0.0 — Inj.

Table 4 shows the percentage of peak areas for the higher molecularweight species in relation to the total peak areas of the eluted speciesfor the syringes of Table 2 incubated for 1 and 3 months at 40° C./75%relative humidity. Each sample was examined in duplicate measurementsunless otherwise noted. All other temperatures (5° C. and 25° C./60%relative humidity) showed no significant increase of higher molecularweight species during storage compared to the starting material.

TABLE 4 Condition Syringe HMWS [%] SD [%] T0 S2 1.1 n.a.*⁾ S6 1.1 n.a.*⁾1M 40° C. S2 10.7 0.1 S6 10.2 0.4 3M 40° C. S2 26.8 0.7 S6 26.3 n.a.*⁾*⁾only single measurement

The generation of HMWS determined by AF4-MALS was highly comparablebetween the two syringes S2 (glass syringe) and S6 (COP) duringincubation at 40° C./75 relative humidity in the period up to 3 months.Both the identities of the higher molecular weight species and thetemperature dependent kinetics were comparable between the two primarypackaging systems.

Example 2.3: SEC Analysis of the Aflibercept Formulation Stored inPrefilled Syringes

The protein samples from the syringes were loaded onto a TSKgelG3000SWXL, (Tosoh, 300×7.8 mm, 5 μm) column to detect high molecularweight species of aflibercept.

The protein was eluted by isocratic elution using 0.02 M sodiumphosphate (pH 6.0) and 0.8 M sodium chloride at a flow rate of 1.0mL/min at 25° C. Eluted species were detected at a wavelength of 214 nmand displayed on a graph showing the concentration of the eluted speciesvs. time. The elution profile showed a main peak with the non-aggregatedprotein and some further peaks of the protein representing highermolecular weight forms of the protein. The area of all peaks wasdetermined. Table 5 shows the percentage of peak area for the aggregatesin relation to the total peak area of the eluted species for thesyringes of Table 2. Each sample was examined in duplicate measurements.

TABLE 5 Condition Syringe HMWS [%] SD [%] T0 S2 2.20 0.01 S6 2.19 0.021M 5° C. S2 2.31 0.01 S6 2.26 0.01 3M 5° C. S2 2.38 0.01 S6 2.36 0.02 2W25° C. S2 2.45 0.01 S6 2.45 0.00 1M 25° C. S2 2.55 0.01 S6 2.53 0.01 3M25° C. S2 3.03 0.01 S6 3.01 0.00 0.5M 40° C.   S2 9.80 0.02 S6 9.76 0.061M 40° C. S2 15.58 0.01 S6 15.49 0.06 3M 40° C. S2 33.71 0.01 S6 33.930.05

The generation of HMWS determined by SEC was highly comparable duringall incubation parameters (temperature, storage time) between the twosyringes S2 (glass syringe) and S6 (COP). Both the identities of thehigher molecular weight species and the temperature dependent kineticswere comparable between the two primary packaging systems.

Example 2.4: Non-Reduced SDS-PAGE Analysis of the AfliberceptFormulation Stored in Prefilled Syringes

By non-reduced SDS-PAGE physical modifications like fragmentation andoligomerization of aflibercept in the different syringe systemsaccording to Table 2 were determined.

The SDS-PAGE was performed under non-reducing conditions in a 4-12%Tris-Glycine gel. Samples were pre-diluted to 0.4 mg/ml with water andfurther diluted to 0.2 mg/ml with SDS sample buffer. The samples wereincubated at 95° C. for 5 min.

After the run the gel was rinsed three times with 100 mL deionized waterand dyed with Coomassie overnight at room temperature. Afterdiscoloration the gel was scanned and analyzed using QuantityOneSoftware.

The running conditions were as follows:

-   -   voltage: 125 V    -   current: 35 mA    -   power: 5 W    -   time: 130 min

Non-reduced SDS-PAGE analysis was performed for samples incubated at alltemperatures listed above for 3 months. Storing the samples at 5° C. didnot lead to significant changes of the banding pattern in all primarypackaging systems, no generation of new impurity bands or significantincrement of existing impurity bands could be detected in both syringematerials over the whole incubation period. Storing the samples at 25°C./60% relative humidity led to stronger impurity bands, the results ofthe non-reduced SDS PAGE analysis of 3 months incubated samples at 40°C./75% relative humidity are shown in FIG. 3.

In the non-reduced SDS-PAGE analysis of all samples incubated for threemonths at 40° C./75 relative humidity bands representing fragments andhigher molecular weight species of aflibercept were visible. Thegeneration of fragments and HMWS during the 3 months incubation washighly comparable in the kinetics and the identity of the impurities inboth primary packaging systems shown in Table 2.

Example 2.5: IEF Analysis of the Aflibercept Formulation Stored inPrefilled Syringes

Isoelectric focusing (IEF) separates different isoforms of afliberceptdue to differences in their isoelectric points because of e.g.deamidation. The ready-to-use IEF gel (Focus Gel (pH 6-11) from Serva,No. 43329.01) contains a pH gradient within the gel. After application,proteins migrate due to their net charge in the pH gradient until theyreach the pH equivalent to their isoelectric point (IEP, IP).

Aflibercept samples were diluted to 0.5 mg/ml with ultrapure water. 10μl thereof equal to 5 μg aflibercept were applied onto the focus gel.Each sample was analyzed as duplicate. After the run the proteins werefixed for 60 minutes in a solution containing 12% (w/v) trichloroaceticacid and 3.5% 5-sulfosalicyl acid dihydrate (w/v), rinsed three timeswith deionized water and dyed with Coomassie overnight at roomtemperature. After discoloration with 20% ethanol the gel was scannedwith a GS 800 densitometer from BioRad and analyzed. Table 6 shows thefocusing conditions:

TABLE 6 Time Power Current Voltage Phase (min) (W) (mA) (V) Pre focusing20 10 50 1000 Sample entrance 30 10 30 500 Isoelectric focusing 90 20 181500 Sharpening 30 25 15 2000

In the IEF no change in the banding pattern of aflibercept compared tothe reference could be detected in all primary packaging systems afterone month storage at all temperatures. After 3 months only samplesincubated at 5° C. and 25° C./60% complied with the reference and showedno alteration in comparison to the starting material. Samples incubatedat 40° C./75% relative humidity showed a comparable shift to acidicspecies in all tested primary packaging materials, there was nodifference with regard to the different primary packaging materialsshown in Table 2.

Example 2.6: Reduced Peptide Mapping Analysis of the AfliberceptFormulation Stored in Prefilled Syringes

By reduced peptide mapping the purity of aflibercept with regard todeamidation and methionine oxidation was analyzed after digestion withtrypsin and liquid chromatography coupled to mass spectrometry (LC-MS)

After reduction and alkylation, the protein was subjected to enzymaticcleavage with trypsin. The resulting peptides were analyzed byRP-UPLC-MS. During chromatography the peptides were eluted by changingthe mobile phase from highly polar (trifluoroacetic acid in water) toless polar (trifluoroacetic acid in acetonitrile) and analyzed by massspectrometry (Xevo G2-XS QTOF). The peptide data was processed andcompared with the theoretical protein sequence and a reference sample todetect oxidations and deamidations.

Syringes shown in Table 2 were analyzed as single measurement after 3months incubation at 5° C., 25° C./60% relative humidity and 40° C./75relative humidity and compared to the starting material t0.

Samples were diluted with denaturation buffer (50 mMTris(hydroxymethyl)aminomethane) to a aflibercept concentration of 1.25mg/mL. 80 μl of the diluted samples were mixed with 10 μl of 0.5%RapiGest (from Waters, solved in 50 mM Tris(hydroxymethyl)aminomethane)and incubated 5 minutes at 95° C. 4.5 μl of 0.02 M DTT (dissolved in 50mM Tris(hydroxymethyl)-aminomethane) were added for reduction andincubated for 30 minutes at 37° C. For aflibercept digestion 5 μl of a 1mg/mL Trypsin solution (solved in 50 mM acetic acid) were added andincubated for further 3 hours at 37° C. The reaction was stopped with 20μl of 2% (v/v) trifluoroacetic acid and an incubation for 30 minutes at37° C. The supernatant was diluted to 0.125 mg/mL with 50 mMTris(hydroxymethyl)-aminomethane for analysis of the peptides.

UPLC Parameters:

The digested protein samples from the syringes were loaded onto anACQUITY UPLC-CSH C-18 column from Waters, 100 mm×2.1 mm, 1.7 μm. 0.25 μgof the digested samples were eluted at 65° C. with a gradient of eluentA (water), eluent B (acetonitrile), eluent C (0.25% trifluoroaceticacid) and D (n-propanol) according to the following Table 7:

TABLE 7 Time Eluent Eluent Eluent Eluent [minutes] A [%] B [%] C [%] D[%] 0.0 89.0 1.0 10.0 0.0 2.5 89.0 1.0 10.0 0.0 5.0 80.0 8.0 10.0 2.050.0 57.5 26.0 10.0 6.5 52.0 0.0 72.0 10.0 18.0 54.0 0.0 72.0 10.0 18.056.0 89.0 1.0 10.0 0.0 60.0 89.0 1.0 10.0 0.0

Method Parameters for Mass Spectrometry:

Ionisation type: ESI Polarity: Positive Analyser mode: SensitivityExperiment type: MS Start Mass: 50 m/z Cone Gas Flow: 30 L/h End Mass:2000 m/z De solvation Gas Flow: 1000 L/h Source Temperature: 120° C.Scan Time: 0.5 s Desolvation Temperature: 450° C. Capillary Voltage: 3.0kV Cone Voltage: 35 V

LockSpray Profile

Reference Compound: Leucine Enkephalin

MS Lock mass: 556.2766 m/z

Scan Time: 0.5 s

Interval: 30 s

4 oxidated methionine residues in aflibercept could be identified in thepeptides (1:T1AS20, 1:T22, 1:T28, 1:T48) and were summed up forevaluation of the total oxidation (see Table 8). 6 deamidation sites ofaflibercept could be identified in the peptides (1:T10_AS12; 1:T11;1:T10_AS12; 1:T12_AS3; 1:T12_AS3; 1:T30_AS12; 1:T30_AS?; 1:T33_AS14) andwere summed up for evaluation of the total deamidation (see Table 8).

TABLE 8 Total Total methionine deamidations Condition Syringe oxidations[%] [%] T0 S2 23.1 35.3 S6 22.5 37.6 3M 5° C. S2 27.7 36.8 S6 22.4 36.93M 25° C. S2 24.5 44.0 S6 23.4 45.3 3M 40° C. S2 25.7 92.0 S6 27.3 90.0

Both syringes shown in Table 2 comprise an identical stability withregard to methionine oxidation and deamidation. Whereas in alltemperature conditions no significant increase of methionine oxidationcould be detected in both syringe materials (glass vs. COP), theincrease of deamidation was temperature dependent. Both syringe systemscomprised a comparable increase of deamidation in the stability program.

From the results shown it is apparent that the stability of anaflibercept formulation of the present invention in a pre-filled plasticsyringe (syringe 6) is at least comparable with the stability in a glasssyringe (syringe 2) under the conditions tested.

Example 3: Second Set of Stability Tests Example 3.1: Sample Preparation

Aflibercept from the EU marketed product Eylea® was transferred by3-step-dialysis into 3 different formulations containing (a) 10 mMhistidine/histidine chloride, 40 mM sodium chloride, 5% (w/v) sucrose,0.03% (w/v) polysorbate 20, pH 6.2 (b) 10 mM histidine/histidinechloride, 40 mM sodium chloride, 5% (w/v) sucrose, 0.03% (w/v)polysorbate 20, pH 6.5 and (c) 10 mM sodium phosphate, 40 mM sodiumchloride, 5% (w/v) sucrose, 0.03% (w/v) polysorbate 20, pH 6.2 as shownin Table 9.

Dialyzed aflibercept was adjusted to 40 mg/mL±10% and stored at 5° C.,25° C./60% relative humidity and 40° C./75% relative humidity for up to3 months in glass vials. Additionally aflibercept in the differentformulations was stressed by five freeze/thaw cycles.

EU marketed product Eylea® was included in the stability program ascontrol sample (d)—see Table 9.

TABLE 9 poly- sodium sorbate Sample aflibercept buffer system sucrosechloride 20 pH (a) 40 mg/mL 10 mM L- 5% 40 mM 0.03% pH histidine/ (w/v)(w/v) 6.2 histidine/HCl (b) 40 mg/mL 10 mM L- 5% 40 mM 0.03% pHhistidine/ (w/v) (w/v) 6.5 histidine/HCl (c) 40 mg/mL 10 mM sodium 5% 40mM 0.03% pH dihydrogen (w/v) (w/v) 6.2 phosphate/ disodium hydrogenphosphate (d) Eylea ®

Afterwards the samples according to Table 9 were analyzed by UV-Vis forprotein concentration, by size exclusion chromatography (SEC) for thepresence of high molecular weight species (HMWS) and byreduced-/non-reduced sodium dodecyl sulfate polyacrylamide gelelectrophoresis (SDS-PAGE) for the presence of fragments and HMWS.Chemical modifications like methionine oxidation and deamidation werequantified by reduced peptide mapping. Alterations of the secondarystructure were monitored by FTIR analysis. The activity of afliberceptin the samples was determined by Potency ELISA and by binding to FcRNvia biolayer interferometry.

Example 3.2: UV-Vis

During the complete stability program no significant changes in proteinconcentration (spectrophotometric quantification at 280 nm; n=3) andappearance (visible particles, change in color) was detected in any ofthe samples.

Example 3.3: Size Exclusion Chromatography

The protein samples of the stability study were loaded onto a TSKgelG3000SWXL, (Tosoh, 300×7.8 mm, 5 μm) column to detect high molecularweight species of aflibercept.

The protein was eluted by isocratic elution using 0.02 M sodiumphosphate (pH 6.0) and 0.8 M sodium chloride at a flow rate of 1.0mL/min at 25° C. Eluted species were detected at a wavelength of 214 nmand displayed on a graph showing the concentration of the eluted speciesvs. time. The elution profile showed a main peak with the non-aggregatedprotein and some further peaks of the protein representing highermolecular weight forms of the protein. The area of all peaks wasdetermined. Table 10 shows the percentage of peak area for theaggregates in relation to the total peak area of the eluted species forthe samples of Table 9. Each sample was examined in duplicatemeasurements and the mean value as well as the standard deviation werecalculated.

TABLE 10 Condition Formulation HMWS [%] SD [%] T0 (a) 1.46 0.00 (b) 1.510.00 (c) 1.50 0.00 (d) 1.49 0.00  1M 5° C. (a) 1.49 0.00 (b) 1.60 0.00(c) 1.49 0.01 (d) 1.57 0.00  2M 5° C. (a) 1.59 0.04 (b) 1.61 0.01 (c)1.51 0.00 (d) 1.62 0.04  3M 5° C. (a) 1.58 0.01 (b) 1.78 0.11 (c) 1.660.00 (d) 1.73 0.03 1M 25° C. (a) 1.63 0.01 (b) 1.88 0.02 (c) 1.81 0.01(d) 2.00 0.01 2M 25° C. (a) 2.03 0.00 (b) 2.20 0.01 (c) 2.13 0.01 (d)2.46 0.01 3M 25° C. (a) 2.30 0.07 (b) 2.78 0.06 (c) 2.32 0.06 (d) 2.790.08 1M 40° C. (a) 13.18 0.00 (b) 12.83 0.00 (c) 13.32 0.00 (d) 13.530.07 2M 40° C. (a) 24.69 0.02 (b) 24.10 0.05 (c) 24.39 0.11 (d) 25.290.10 3M 40° C. (a) 29.77 0.13 (b) 28.40 0.33 (c) 29.19 0.34 (d) 30.270.20

The generation of HMWS as determined by SEC was comparable between thedifferent formulations for all incubation parameters (temperature,storage time). Both the identity of the higher molecular weight speciesand the temperature dependent kinetics were comparable between thedifferent samples.

Example 3.4: Non-Reduced SDS-PAGE

The non-reduced SDS-PAGE was performed using the conditions described inExample 2.4 with samples stored at each of the storage temperatures for3 months.

Storing the samples at 5° C. or 25° C./60% relative humidity did notlead to significant changes of the banding pattern in all formulations.In particular, no new impurity bands or a significant increase ofexisting impurity bands could be detected in all samples shown in Table9 over the whole incubation period. Storing the samples at 40° C./75%relative humidity led to stronger impurity bands compared to the samplesstored at lower temperatures. The results of the non-reduced SDS PAGEanalysis of samples incubated at 40° C./75% relative humidity for threemonths are shown in FIG. 4. Every sample was evaluated as duplicatemeasurement in the gel.

In the non-reduced SDS-PAGE analysis of all samples incubated for threemonths at 40° C./75% relative humidity bands representing fragments andhigher molecular weight species of aflibercept were visible. Thegeneration of fragments and HMWS during the 3 months incubation wascomparable in the kinetics and the identity of the impurities betweenall tested formulations shown in Table 9.

Example 3.5: Reduced SDS-PAGE

By reduced SDS-PAGE physical modifications such as fragmentation andoligomerization of aflibercept in the different formulations accordingto Table 9 were determined.

The SDS-PAGE was performed under reducing conditions in a 4-12%Tris-Glycine gel. Samples were pre-diluted to 0.4 mg/ml with water andfurther diluted to 0.2 mg/ml with SDS sample buffer containing DTT. Thesamples were incubated at 95° C. for 5 min.

After the run the gel was rinsed three times with 100 mL deionized waterand dyed with Coomassie overnight at room temperature. Afterdiscoloration the gel was scanned and analyzed using QuantityOneSoftware.

The miming conditions were as follows:

-   -   voltage: 125 V    -   current: 35 mA    -   power: 5 W    -   time: 200 min

The non-reduced SDS-PAGE was performed with samples stored at each ofthe storage temperatures for 3 months.

Storing the samples at 5° C. or 25° C./60% relative humidity did notlead to significant changes of the banding pattern in all formulations.In particular, no generation of new impurity bands or a significantincrease of existing impurity bands could be detected in all samplesshown in Table 9 over the whole incubation period. Storing the samplesat 40° C./75% relative humidity led to stronger impurity bands comparedto the samples stored at lower temperatures. These impurity bands werecomparable between all tested formulations shown in Table 9 in both thekinetics and the identity of the impurities. The results of the reducedSDS PAGE analysis of samples incubated for three months at 40° C./75%relative humidity are shown in FIG. 5.

Example 3.6: FTIR

FTIR (Fourier transform infrared spectroscopy) spectroscopy providesinformation on the secondary structure of proteins and works byexcitation of a sample with infrared radiation and detection of thewavelengths absorbed by the protein.

Each protein has a characteristic set of absorption bands in itsinfrared spectrum. Characteristic bands found in the infrared spectra ofproteins and polypeptides include the Amide I and Amide II region. Thesearise from the peptide bonds that link the amino acids in the proteinbackbone. The Amide I band was evaluated in this assay to monitor thesecondary structure components alpha helices and beta sheets.

Samples from the stability studies were diluted with their correspondingformulation without aflibercept (placebo formulation) to a concentrationof 10 mg/mL aflibercept and analyzed by a FTIR Tensor27 from BrukerOptics in an AquaSpec cell from Micro Biolytics. Data analysis wasperformed with Opus 6.5 software (Bruker Optics). 10 measurements wereperformed with an injection volume of 2.0 μl and the second derivativespectra were analyzed in the Amide I absorption area from 1700-1600cm⁻¹. Analysis of the formulations without aflibercept served asbackground measurements and the signals were subtracted from the proteinspectra.

Device setting for FTIR

Resolution: 4 cm⁻¹

Sample Scan Time: 30 scans

Background Scan Time: 30 scans

Result spectrum: Absorbance

Source Setting: MIR

Beamsplitter: KBr

Detector Setting: LN-MCT Photovoltaic 12 H

Scanner Velocity: 20 kHz

Water batch: 25° C.

The analysis of the aflibercept containing formulations shown in Table 9by FTIR with regard to secondary structure did not reveal any alterationduring the complete stability program. All samples showed a comparableconstant percentage of about 10% alpha helicesand 40% beta-sheets.

Example 3.7: Reduced Peptide Mapping

By reduced peptide mapping the purity of aflibercept with regard toasparagine deamidation and methionine oxidation was analyzed for allformulations of Table 9 which were stored at different temperatures forthree months or subjected to five freeze/thaw cycles. The conditions forthe analysis were the same as those used in example 2.6.

5 oxidated methionines in aflibercept could be identified (AA 20; AA163; AA 192, AA 237, AA 413) and were summed up for evaluation of thetotal methionine oxidation (see Table 11). 5 deamidations of asparaginecould be identified (AA 84; AA 91; AA 99; AA 271; AA 300) and weresummed up for evaluation of the total deamidation (see Table 11)

TABLE 11 Total Total methionine deamidations Condition Formulationoxidations [%] [%] T0 (a) 22.1 33.3 (b) 21.3 29.7 (c) 25.1 31.0 (d) 24.030.8 3M 5° C.  (a) 23.6 32.4 (b) 21.8 31.2 (c) 24.1 32.4 (d) 23.3 31.63M 25° C. (a) 24.3 40.0 (b) 25.2 46.2 (c) 27.1 39.2 (d) 23.8 37.9 3M 40°C. (a) 27.4 87.6 (b) 24.6 102.2 (c) 29.5 87.1 (d) 25.0 102.2 5 × F/T (a)21.1 32.4 (b) 19.1 30.5 (c) 24.4 34.1 (d) 22.5 33.6

The formulations shown in Table 9 showed a comparable stability withregard to methionine oxidation and deamidation of asparagine. Whereas atall temperature conditions only slight increases of methionine oxidationin the different formulations were detected, the increase ofdeamidations was significantly temperature-dependent. Allaflibercept-containing formulations of Table 9 showed a comparableincrease of deamidations when stored for 3 months at 25° C./60% relativehumidity or 40° C./75% relative humidity. Freeze/thaw did not show aninfluence on both methionine oxidation and asparagine deamidations inall tested formulations.

Example 3.8: Relative Potency

The relative potency of aflibercept was determined by an ELISA (enzymelinked immunosorbent assay) which is based on the binding of afliberceptto Vascular Endothelial Growth Factor (VEGF).

All test samples were diluted to an assay concentration of 1.7 pMaflibercept with StartingBlock (PBS) Blocking Buffer (Thermo Fisher, No.37538), mixed with VEGF-A165 (2.5 ng/mL assay concentration) andincubated overnight at 4° C. 100 μl of the samples were transferred to amicrotiter plate coated with an anti-VEGF antibody. Non-neutralized VEGFbound to the coated anti-VEGF antibody in the microtiter-well, whilecomplexes of aflibercept/VEGF were removed by three washing steps (each300 μl) with PBS/0.1% (w/v) polysorbate 20. The microtiter plate wassealed with an adhesive foil and incubated for 15 minutes at roomtemperature with adequate shaking. After additional washing steps (3×300μl PBS/0.1% (w/v) polysorbate 20) the binding of VEGF was detected byaddition of a biotinylated polyclonal anti-VEGF 165 antibody (100 μl ofa 200 μg/mL solution), sealing, incubation for further 120 minutes inthe dark and visualized using a Streptavidin-HRP (Horseradishperoxidase) conjugate via the oxidation of3,3′,5,5′-tetramethylbenzidine (TMB). The colorimetric reaction wasstopped after 20 minutes incubation in the dark by adequate shaking withsulphuric acid, the absorption was measured at a wavelength of 450 nmwith a Fluorescence reader for microtiter plates (Tecan Infinite M200Pro) and compared to an aflibercept standard.

All test samples were analyzed as duplicates.

During the 3 months incubation at 5° C. and 25° C./60% relative humidityno significant loss of the relative potency could be detected in allformulations shown in Table 9 compared to the starting material. Alsofreeze/thaw cycles did not influence the relative potency of afliberceptin each of the tested formulations. Incubation at 40° C./75% relativehumidity for three months led to a comparable decrease of the relativepotency in all formulations.

Example 3.9: Binding to FcRN

The evaluation of the kinetic parameters (KD, k_(on), k_(dis)) usingBio-Layer Interferometry technology ((Pall-FortéBIO Octet RED 96) forthe interaction between the neonatal Fc receptor (FcRn) and afliberceptin the formulations shown in Table 9 during the stability assays showeda similar picture.

Bio-Layer Interferometry (BLI) is a label-free technology for measuringbiomolecular interactions. It is an optical analytical technique thatanalyzes the interference pattern of white light reflected from twosurfaces: a layer of immobilized protein on the biosensor tip and aninternal reference layer. The binding between HIS-tagged neonatal Fcreceptor (FcRn, No. CT009-H08H from Sino Biological Inc.) immobilized onthe surface of Ni-NTA biosensors coated with nickel-charged Tris-NTA(No. 18-5101 from Pall-FortéBIO) and aflibercept produces an increase inoptical thickness at the biosensor tip, which results in a wavelengthshift, which is a direct measure of the change in thickness of thebiological layer and provides the ability to determine bindingspecificity and rates of association and dissociation.

Samples from Table 9 were analyzed after 3 months incubation at 5° C.and 40° C./75% relative humidity and compared to the starting material.The samples were diluted with their corresponding formulation withoutaflibercept (placebo formulation) to 10 mg/mL aflibercept and furtherdiluted to 0.704 μg/mL, 0.352 μg/mL and 0.176 μg/mL aflibercept withkinetic buffer (DPBS/0.05% (w/v) BSA/0.02% (w/v) polysorbate 20/0.5 Msodium chloride, pH 6.0). Ni-NTA biosensors were hydrated with 200 μlkinetic buffer.

Each well was filled with 200 μl of kinetic buffer, ligand solution andanalyte solution according to following scheme in Table 12:

TABLE 12 Ligand Baseline 3 + Aflibercept Baseline [mg/mL] Baseline 2Dissociation [mg/mL] 1/7 2/8 3/9 4/10 5/11 A Kinetic buffer 1.0 Kineticbuffer Kinetic buffer 0.704 B Kinetic buffer 1.0 Kinetic buffer Kineticbuffer 0.352 C Kinetic buffer 1.0 Kinetic buffer Kinetic buffer 0.176 DKinetic buffer 1.0 Kinetic buffer Kinetic buffer Kinetic buffer

Kinetic buffer was pipetted into row D instead of analyte to serve asreference.

Measurement Parameters:

-   -   Plate temperature: 30° C.    -   Shaking speed: 1000 rpm    -   Acquisition rate: Standard kinetics (5.0 Hz averaging by 20)

The signal of the sample was recorded according to Table 13:

TABLE 13 Time Sample No. Step [sec.] column 1 Baseline 300 1/7 2 Loading600 2/8 3 Baseline 2 150 3/9 4 Baseline 3 150  4/10 5 Assiciation 400 5/11 6 Dissociation 2400  4/10

The data were analyzed with the Octet Data Analysis software resultingin the kinetic parameters K_(D), k_(on) and k_(dis) values.

TABLE 14 Condition Formulation K_(D) k_(on) k_(dis) T0 (a) 1.18E−10M5.56E+05 1/M*s 6.56E−05 1/s (b) 1.06E−11M 6.13E+05 1/M*s 6.47E−05 1/s(c) 1.07E−10M 6.05E+05 1/M*s 6.44E−05 1/s (d) 9.93E−11M 5.60E+05 1/M*s5.56E−05 1/s 3M 5° C. (a) 9.80E−11M 5.96E+05 1/M*s 5.84E−05 1/s (b)9.23E−11M 6.19E+05 1/M*s 5.71E−05 1/s (c) 1.03E−10M 6.10E+05 1/M*s6.28E−05 1/s (d) 9.68E−11M 6.31E+05 1/M*s 6.11E−05 1/s 3M 40° C. (a)9.05E−11M 4.54E+05 1/M*s 4.11E−05 1/s (b) 5.91E−11M 4.83E+05 1/M*s2.86E−05 1/s (c) 5.91E−11M 4.59E+05 1/M*s 2.71E−05 1/s (d) 6.09E−11M4.93E+05 1/M*s 3.00E−05 1/s

The kinetic parameters (K_(D), k_(on) and k_(off)) were similar to thestarting conditions after 3 months of incubation at 5° C. for allformulations. Storing the samples for 3 months at 40° C./75% relativehumidity led to a decrease of K_(D) which was mainly due to thedecreased dissociation rate k_(dis). This decrease was comparable forall tested formulations of Table 9.

In summary, no significant differences between the formulations shown inTable 9 could be detected both with regard to physical and chemicalstability when stored for 3 months at 5° C., 25° C./60% relativehumidity and 40° C./75% relative humidity or treated by freeze/thawcycles, also the trend in biological activity did not differ between theformulations. From the results shown it is apparent that the stabilityof aflibercept in the histidine-based formulations of the presentinvention (a and b) is at least comparable with the stability in theformulation of the phosphate-buffered formulation (c) or the EU marketedEylea® under the conditions tested.

1. A method of treating an intraocular neovascular disease comprisingadministering a liquid pharmaceutical composition comprising a) ahistidine containing buffer, b) a non-ionic surfactant, c) a VEGFantagonist, d) an inorganic salt, and e) a carbohydrate.
 2. The methodof claim 1, wherein the pH of the composition is between 6.0 and 6.5,preferably between 6.2 and 6.5.
 3. The method of claim 1, wherein thehistidine-containing buffer is L-histidine/histidine hydrochloride. 4.The method of claim 1, wherein the histidine-containing buffer ispresent in a concentration of from 1 mM to 40 mM, preferably of 10 mM.5. The method of claim 1, wherein the non-ionic surfactant ispolysorbate
 20. 6. The method of claim 1, wherein the non-ionicsurfactant is present in a concentration of from 0.01 to 0.08% (w/v),preferably of 0.03% (w/v).
 7. The method of claim 1, wherein theinorganic salt is NaCl.
 8. The method of claim 1, wherein the inorganicsalt is present in a concentration of from 20 to 100 mM.
 9. The methodof claim 1, wherein the VEGF antagonist is an anti-VEGF antibody or anantigen-binding fragment of such antibody or a VEGF receptor fusionprotein.
 10. The method of claim 1, wherein the VEGF antagonist isaflibercept or ranibizumab.
 11. The method of claim 1, wherein the VEGFantagonist is present in a concentration of 6 to 45 mg/ml.
 12. Themethod of claim 1, wherein the carbohydrate is sucrose.
 13. The methodof claim 1, wherein the carbohydrate is present in a concentration of3-20% (w/v).
 14. A method of treating an intraocular neovascular diseasecomprising administering a liquid pharmaceutical composition consistingof histidine hydrochloride/L-histidine, polysorbate 20, NaCl,aflibercept, sucrose and water and having a pH of 6.2 or 6.5.
 15. Themethod of claim 14, wherein the liquid pharmaceutical compositionconsists of 10 mM histidine hydrochloride/L-histidine, 0.03% polysorbate20 (w/v), 40 mM NaCl, 40 mg/ml aflibercept, 5% sucrose and water and hasa pH of 6.2 or 6.5.
 16. The method of claim 1, wherein the intraocularneovascular disease is age-related macular degeneration (AMD), visualimpairment due to diabetic macular oedema (DME), visual impairment dueto macular oedema secondary to retinal vein occlusion (branch RVO orcentral RVO), or visual impairment due to choroidal neovascularisation(CNV) secondary to pathologic myopia.
 17. The method of claim 1, whereinthe pharmaceutical composition is present in a prefilled syringe. 18.The method of claim 6, wherein the non-ionic surfactant is present in aconcentration of 0.03% (w/v).
 19. The method of claim 8, wherein theinorganic salt is present in a concentration of 40 mM.
 20. The method ofclaim 13, wherein the carbohydrate is present in a concentration of 5%(w/v).